1. Field of the Invention
The present invention relates to a reaction velocity measuring apparatus for rapid and accurate measurement of the velocity of chemical reactions.
2. Description of the Prior Art
The principle of the measurement of velocity of chemical reaction is explained in the following with particular reference, as an example, to an enzymatic reaction.
It is already known to determine the amount of enzyme indirectly from the reaction velocity per unit time or enzyme activity by measuring the reaction velocity of a reaction catalyzed by said enzyme. Under certain conditions, i.e. under suitable temperature conditions (under 30.degree. C, for example) with a suitably low enzyme concentration and a suitably high substrate concentration, each molecule of enzyme independently reacts with the substrate with a velocity proportional to the concentration of enzyme. In case the reaction velocity is proportional to the amount of enzyme and does not depend on time, the enzymatic reaction proceeds at a constant velocity. Stated differently the amount of reaction product stands in a linear relationship with time, signifying that the light absorption coefficient becomes proportional to time.
The measurement of the reaction velocity of enzyme is usually achieved by measuring the difference of light absorbance in one minute while such proportional relationship stands, and the IU, i.e. the amount of enzyme per one liter is defined by the following equation: EQU IU = .DELTA.A.multidot.K
wherein .DELTA.A represents the difference of light absorbance in one minute, and K is a constant (hereinafter referred to as K factor).
In the prior art the velocity of an enzymatic reaction is measured by recording the light absorbance on a recorder, reading the difference of light absorbance in one minute within a range where such linear relationship is visually observable and multiplying said difference by said K factor manually or by measuring light absorbance at suitable time intervals, electrically subtracting and converting thus obtained values to obtain the difference in one minute, and multiplying said difference by said K factor.
In case of determining the enzyme activity from the change of light absorbance in the course of a reaction, the period required for such determination is represented by (number of samples) .times. (time of measurement necessary for one sample). On the other hand the colorimetry method wherein the reaction velocity is calculated from the light absorbance in the chemical equilibrium state after the completion of chemical reaction requires a period represented by (time necessary for reaction) + (number of samples) .times. (period of measurement necessary for one sample) since in this case plural samples are simultaneously subjected to reaction and then to measurements after the completion of reaction. The former requires a shorter time and is therefore advantageous if the number of samples is limited, but takes several times longer period in comparison with the latter which requires only several seconds for the measurement of each sample, if a considerably large number of samples is to be measured. With respect to the accuracy of measurement, however, the latter is inferior since the measurement is realized in a range where the light absorbance is not linearly correlated with the reaction velocity. For this reason the former method is more adequate for a more accurate measurement of the enzyme activity. With further respect to the former method, the usually employed way with electric subtraction is realized by the measurement of light absorbance A at time t.sub.1 followed by that of light absorbance A' at time t.sub.2 to determine a ratio (A.sub.2 - A.sub.1)/(t.sub.2 - t.sub.1).
In this manner the measuring apparatus is not in operation between two measurements at t.sub.1 and t.sub.2, and has therefore considerable idle time. It has therefore been proposed to utilize such idle time between t.sub.1 and t.sub.2 for the measurements of other samples thereby improving the efficiency of apparatus and increasing the number of treated samples. In this case the sample measured at time t.sub.1 has to be returned to the original position at t.sub.2 since a sample has to be subjected to two measurements. For this purpose there have been proposed devices in which plural samples are arranged in a reciprocating rack or arranged in circular positions. However such devices are inadvantageous as the number of samples accomodated is inevitably limited. More specifically the devices of this kind become inevitably large in dimensions, if the number of samples is increased, and require complicated electric circuitry as the memory circuits explained later are required in same number as the samples. Thus, there have to be frequent interruptions of operations for the replacement of measured samples with unmeasured ones.
Furthermore, because two measurements are effected with time interval t.sub.1 - t.sub.2, there would be variation in the optical source or drifts of electrical portions such as amplifier, which adversely effects measurement accuracy. It is better to adopt double beam method for eliminating such drawback, which method can eliminate such influence caused by fluctuation in the light source or by a back ground condition which is constant between times t.sub.1 - t.sub.2 as contamination of the sample cell for measuring the difference of absorbance. However, it is impossible to eliminate an undesirable influence caused by back ground such as bubbles in the cell, which varies between the period t.sub.1 - t.sub.2. For effecting such elimination it is necessary to adopt spectrophotometry at two wavelengths, which enables to eliminate back grounds at time t.sub.1 and time t.sub.2 respectively so that measurements can be made with accuracy even if there occur any variations in back grounds between the time period t.sub.1 - t.sub.2.
In the following there is given a detailed description on the measurement of reaction velocity by spectrophotometry at two wavelengths.
The absorbances A.sub.1 and A.sub.2 at wavelengths .lambda..sub.1 and .lambda..sub.2 of a sample at a time t.sub.1 can be expressed by the following equations: ##EQU1## wherein I.lambda..sub.10 (t.sub.1): incident light of wavelength .lambda..sub.1
I.lambda..sub.20 (t.sub.1): incident light of wavelength .lambda..sub.2 PA1 I.lambda..sub.1 (t.sub.1): transmitted light of wavelength .lambda..sub.1 PA1 I.lambda..sub.2 (t.sub.1): transmitted light of wavelength .lambda..sub.2 PA1 K.sub.1 : absorption coefficient at .lambda..sub.1 PA1 K.sub.2 : absorption coefficient at .lambda..sub.2 PA1 c: sample concentration PA1 x: light path length PA1 b.sub.1, b.sub.2 : back grounds at .lambda..sub.1, .lambda..sub.2.
It can be assumed that b.sub.1 = b.sub.2 if the wavelengths .lambda..sub.1 and .lambda..sub.2 are selected mutually close.
Similarly following equations stand at a time t.sub.2 : ##EQU2## The difference of absorbances at .lambda..sub.1 and .lambda..sub.2 at time t.sub.1 is obtained as follows: ##EQU3## Similarly the difference of absorbance .DELTA.A (t.sub.2) at time t.sub.2 is: ##EQU4## If the fluctuation of light source is small between times t.sub.1 and t.sub.2, there stands: ##EQU5## Thus the difference of absorbances at t.sub.1 and t.sub.2 can be expressed as follows: ##EQU6## and is therefore proportional to the change .DELTA.c of concentration of the sample between times t.sub.1 and t.sub.2. Thus, by multiplying a factor K' = k.multidot.[K.sub.1 /, (K.sub.2 - K.sub.1)] the amount IU of the enzyme is represented as follows: EQU IU = K' {.DELTA.A(t.sub.2) - .DELTA.A(t.sub.1)} = K.multidot.K.sub.1 .multidot..DELTA.c.multidot.x = K .multidot. .DELTA.A (8)
in the spectrophotometric method with two wavelengths it is necessary to use a same optical system for the measurements at t.sub.1 and t.sub.2 since there will be a difference in wavelength characteristics if different optical systems are employed.